Preparation of inactivated viral vaccines

ABSTRACT

Vaccines employing inactivated viruses having improved retention of antigenic characteristics are prepared by psoralen-inactivation of the live virus in a non-oxidizing atmosphere. By excluding oxygen and other oxidizing species from the inactivation medium, degradation of the antigen characteristics resulting from irradiation with ultraviolet light is largely prevented. The resulting inactivated viruses are employed in vaccine preparations for the inoculation of susceptible hosts to inhibit viral infection.

This application is a continuation-in-part of application Ser. No.563,939, filed on Dec. 20, 1983, now U.S. Pat. No. 4,545,987, andapplication Ser. No. 592,661, filed on Mar. 23, 1984, abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the preparation of inactivated viralvaccines. More particularly, the invention relates to psoraleninactivation of viral particles under conditions which limit antigenicdegradation of the viral particles caused by the inactivation.

Vaccination against both bacterial and viral diseases has been one ofthe major accomplishments of medicine over the past century. Whileeffective vaccines have been developed for a large number of diseases,development of safe and effective vaccines for a number of otherdiseases remains problematic. The use of inactivated or killed microbialagents as a vaccine, although generally safe, will not always beeffective if the immunogenic characteristics of the agent are altered.Indeed, the preferential degradation of certain antigens on theinactivated microorganisms might produce an immune response which allowsfor an immunopathological response when the host is later challengedwith the live microorganism. In contrast, the preparation of live,attenuated microbial agents as a vaccine will often provide improvedimmunologic reactivity, but increases the risk that the vaccine itselfwill be infectious, e.g., as a result of reversion, and that theorganism will be able to propagate and provide a reservoir for futureinfection.

Thus, one must generally choose between improved effectiveness andgreater degree of safety when selecting between the viral inactivationand viral attenuation techniques for vaccine preparation. The choice isparticularly difficult when the virus is resistant to inactivation andrequires highly rigorous inactivation conditions which are likely todegrade the antigenic characteristics.

It is therefore desirable to provide improved methods for inactivatingviruses, which methods are capable of inactivating even the mostresistant viruses under conditions which do not substantially degradethe antigenic structure of the viral particles. In particular, theinactivated viruses should be useful as vaccines and free from adverseside effects at the time of administration as well as upon subsequentchallenge with the live virus.

2. Description of the Prior Art

The reactivity of psoralen derivatives with viruses has been studied.See, Hearst and Thiry (1977) Nuc. Acids Res. 4:1339-1347; and Talib andBanerjee (1982) Virology 118:430-438. U.S. Pat. No. 4,124,598 and4,196,281 to Hearst et al. suggest the use of psoralen derivatives toinactivate RNA viruses, but include no discussion of the suitability ofsuch inactivated viruses as vaccines. U.S. Pat. No. 4,169,204 to Hearstet al. suggests that psoralens may provide a means for inactivatingviruses for the purpose of vaccine production but presents noexperimental support for this proposition. European patent application 0066 886 by Kronenberg teaches the use of psoralen inactivated cells,such as virus-infected mammalian cells, for use as immunologicalreagents and vaccines. Hanson (1983) in: Medical Virology II, de la Mazaand Peterson, eds., Elsevier Biomedical, New York, pp. 45-79, reportsstudies which have suggested that oxidative photoreactions betweenpsoralens and proteins may occur.

SUMMARY OF THE INVENTION

The present invention provides for the production offurocoumarin-inactivated viral vaccines under conditions whichsubstantially preserve the antigenic characteristics of the inactivatedviral particles. It has been recognized by the inventors herein that theinactivation of viruses by exposure to ultraviolet radiation in thepresence of furocoumarin compounds can degrade the antigenic structureof the viral particle. While such degradation can be limited byemploying less rigorous inactivation conditions, certain recalcitrantviruses require relatively harsh inactivation conditions in order toassure that all residual infectivity is eliminated. The inactivationconditions required to eliminate substantially all infectivity in suchrecalcitrant viruses can so degrade the viral particle that it isunsuitable for use as the immunogenic substance in a vaccine. Even ifthe degradation is not so complete, partial degradation of the antigeniccharacteristics may render the vaccine less effective than would bedesirable. That is, the vaccine may require higher concentrations of theinactivated viral particles in each inoculation, and/or the vaccinationprogram may require additional inoculations in order to achieveimmunity.

According to the present invention, vaccines are prepared by treatmentwith furocoumarins and long wavelength ultraviolet (UVA) light underconditions which limit the availability of oxygen and other reactive,particularly oxidizing, species. It has been found that such conditionsallow for the inactivation of even recalcitrant viral particles withoutsubstantial degradation of the antigenic characteristics of thoseparticles. Thus, viruses which have heretofore been resistant tofurocoumarin-inactivation may now be inactivated without loss of thedesired immunogenicity, and viruses which have previously beensuccessfully inactivated may now be inactivated under conditions whichbetter preserve their antigenic characteristics, making them moreefficient immunogenic substances for use in vaccines.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

According to the present invention, vaccines useful for the inoculationof mammalian hosts, including both animals and humans, against viralinfection are provided. The vaccines are prepared by inactivation oflive virus in an inactivation medium containing an amount of aninactivating furocoumarin sufficient to inactivate the virus uponsubsequent irradiation with long wavelength ultraviolet radiation.Degradation of the antigenic characteristics of the live virus isreduced or eliminated by limiting the availability of oxygen and otheroxidizing species in the inactivation medium. Suitable vaccines may beprepared by combining the inactivated viruses with aphysiologically-acceptable carrier, typically an adjuvant, in anappropriate amount to elicit an immune response, e.g., the production ofserum neutralizing antibodies, upon subsequent inoculation of the host.

The present invention is suitable for producing vaccines to a widevariety of viruses, including human viruses and animal viruses, such ascanine, feline, bovine, porcine, equine, and ovine viruses. The methodis suitable for inactivating double stranded DNA viruses,single-stranded DNA viruses, double-stranded RNA viruses, andsingle-stranded RNA viruses, including both enveloped and non-envelopedviruses. The following list is representative of those viruses which maybe inactivated by the method of the present invention.

    ______________________________________                                        Viruses which may be inactivated                                                           Representative Viruses                                           ______________________________________                                        dsDNA                                                                         Adenoviruses   Adenovirus, canine adenovirus 2                                Herpesviruses  Herpes simplex viruses,                                                       Feline Herpes I                                                Papovaviruses  Polyoma, Papilloma                                             Poxviruses     Vaccinia                                                       ssDNA                                                                         Parvovirus     Canine parvovirus, Feline                                                     panleukopenia                                                  dsRNA                                                                         Orbiviruses    Bluetongue virus                                               Reoviruses     Reovirus                                                       ssRNA                                                                         Calicivirus    Feline calicivirus                                             Coronavirus    Feline infectious peritonitis                                  Myxovirus      Influenza virus                                                Paramyxovirus  Measles virus, Mumps virus,                                                   Newcastle disease virus,                                                      Canine distempter virus,                                                      Canine parainfluenza 2 virus                                   Picornavirus   Polio virus, Foot and mouth                                                   disease virus                                                  Retrovirus     Feline leukemia virus, Human                                                  T-cell lymphoma virus, types                                                  I, II and III                                                  Rhabdovirus    Vesicular stomatitis virus,                                                   rabies                                                         Togavirus      Yellow fever virus, Sindbis                                                   virus, Encephalitis virus                                      ______________________________________                                    

Of particular interest are those viruses for which conventional vaccineapproaches have been unsuccessful or marginally successful. For suchviruses, inactivation procedures which are sufficiently rigorous toassure the total loss of infectivity often result in partial or completedestruction of the antigenic characteristics of the virus. With suchloss of antigenic characteristics, the viruses are incapable ofeliciting a protective immunity when administered to a susceptible host.While it would be possible to utilize less rigorous inactivationconditions in order to preserve the antigenic integrity of the virus,this approach is not desirable since it can result in incompleteinactivation of the virus.

In preparing the subject vaccines, sufficient amounts of the virus to beinactivated may be obtained by growing seed virus in a suitablemammalian cell culture. Seed virus, in turn, may be obtained byisolation from an infected host. Suitable mammalian cell culturesinclude primary or secondary cultures derived from mammalian tissues orestablished cell lines such as Vero cells, monkey kidney cells, BHK21hampster cells, LMTK³¹ cells, and other cells permissive for the desiredvirus and which may be grown in vitro as monolayer or suspensioncultures. The cell cultures are grown to approximately 80% saturationdensity, and infected with the virus at a low multiplicity of infection(MOI), usually between about 0.05 and 0.005, preferably at about 0.01.After adsorbing the viral inoculum to the cells by incubation for alimited period of time at a temperature in the range from 35° C. to 40°C., an appropriate growth or maintenance medium is added. The cells arefurther incubated at about the same temperature, in the presence ofabout 5% carbon dioxide in air, until a sufficient amount of virus hasbeen produced.

The growth and maintenance medium will usually be a conventionalmammalian cell culture medium, such as Eagle's Minimum Essential Mediumor Medium 199, usually supplemented with additives such as brothprepared from dehydrated standard microbial culture media, fetal bovineserum, fetal calf serum, or the like.

The furocoumarins useful for inactivation are primarily illustrated bythe class of compounds referred to as psoralens, including psoralen andsubstituted psoralens where the substituents may be alkyl, particularlyhaving from one to three carbon atoms, e.g., methyl; alkoxy,particularly having from one to three carbon atoms, e.g., methoxy; andsubstituted alkyl having from one to six, more usually from one to threecarbon atoms and from one to two heteroatoms, which may be oxy,particularly hydroxy or alkoxy having from one to three carbon atoms,e.g., hydroxy methyl and methoxy methyl, or amino, including mono- anddialkyl amino or aminoalkyl, having a total of from zero to six carbonatoms, e.g., aminomethyl. There will be from 1 to 5, usually from 2 to 4substituents, which will normally be at the 4, 5, 8, 4' and 5'positions, particularly at the 4' position. Illustrative compoundsinclude 5-methoxypsoralen; 8-methoxypsoralen (8-MOP); 4,5',8-trimethylpsoralen (TMP); 4'-hydroxymethyl-4,5', 8-trimethylpsoralen(HMT); 4'-aminomethyl-4,5', 8-trimethylpsoralen (AMT); 4-methylpsoralen;4,4'-dimethylpsoralen; 4,5'-dimethylpsoralen; 4', 8 -dimethylpsoralen;and 4'-methoxymethyl-4,5', 8-trimethylpsoralen. Of particular interestare AMT and 8-MOP.

The furocoumarins may be used individually or in combination. Each ofthe furocoumarins may be present in amounts ranging from about 0.01μg/ml to 1 mg/ml, preferably from about 0.5 μg/ml to 100 μg/ml, therenot being less than about 1 μg/ml nor more than about 1 mg/ml offurocoumarins.

In carrying out the invention the furocoumarin(s), in an appropriatesolvent which is substantially inert and sufficiently non-polar to allowfor dissolution of the furocoumarin(s), are combined with the viralsuspension, conveniently a viral suspension in an aqueous bufferedmedium, such as used for storage. The amount of virus will generally beabout 1×10⁶ to 10¹¹, more usually about 1×10⁷ to 10⁹ and preferablyabout 1×10⁸ to 5×10⁸ pfu/ml The furocoumarin(s) will be at aconcentration of about 0.001 mg/ml to 0.5 mg/ml, more usually about 0.05mg/ml to 0.2 mg/ml. The amount of solvent which is used to dissolve thefurocoumarin will be sufficiently small so as to readily dissolve in theaqueous viral suspension.

Although viral inactivation according to the present invention willnormally be carried out in an inactivation medium as just described, insome cases it may be desirable to introduce furocoumarins to the virusby addition to a cell culture medium in which the virus is grown. Theinactivation is then carried out by separating the live viral particlesfrom the culture medium, and exposure of the particles to ultravioletlight in an inactivation medium which may or may not contain additionalfurocoumarins. This method of inactivation is useful where the virus isresistant to inactivation when the furocoumarin is added to theinactivation medium only.

When employing furocoumarins with limited aqueous solubility, typicallybelow about 50 g/ml, it has been found useful to add an organic solvent,such as dimethyl sulfoxide (DMSO), ethanol, glycerol, polyethyleneglycol (PEG) or polypropylene glycol, to the aqueous treatment solution.Such furocoumarins having limited solubility include 8-MOP, TMP, andpsoralen. By adding small amounts of such organic solvents to theaqueous composition, typically in the range from about 1 to 25% byweight, more typically from about 2 to 10% by weight, the solubility ofthe furocoumarin can be increased to about 200 μg/ml, or higher. Suchincreased furocoumarin concentration may permit the use of shorterirradiation times. Also, inactivation of particularly recalcitrantmicroorganisms may be facilitated without having to increase the lengthor intensity of ultraviolet exposure, and the addition of an organicsolvent may be necessary for inactivation of some viruses withparticular furocoumarins. The ability to employ less rigorousinactivation conditions is of great benefit in preserving theantigenicity of the virus during inactivation.

At times, it may be desirable to employ organic solvents, particularlyDMSO, with all furocoumarins regardless of solubility. For somemicroorganisms, particularly viruses having tight capsids, the additionof the organic solvent may increase the permeability of the outer coator membrane of the microorganism. Such increase in permeability wouldfacilitate penetration by the furocoumarins and enhances theinactivation of the microorganism.

The furocoumarin may be added to the viral suspension in a singleaddition or in multiple additions, where the virus is irradiated betweenadditions, or may be added continuously during the entire treatmentperiod, or a portion thereof. Usually, the number of additions will befrom about 1 to 50, more usually from about 10 to 40, and preferablyfrom about 2 to 4. The total amount of furocoumarin which will be addedwill be sufficient to provide a concentration of at least about 0.01mg/ml to about 1 mg/ml, usually not more than about 0.75 mg/ml andpreferably not more than about 0.5 mg/ml. Since a substantial proportionof the furocoumarin will have reacted with the nucleic acid betweenadditions, the total concentration of furocoumarin in solution willgenerally not exceed about 0.1 mg/ml. In cases where the furocoumarin(s)employed are particularly unstable, it may be beneficial to add thefurocoumarin solution continuously during the irradiation procedure.

In order to preserve the antigenic characteristics of the virus,irradiation is carried out in the substantial absence of oxygen andother oxidizing species. This is particularly important when employingpsoralens that generate more singlet oxygen on a molar basis. Forexample, AMT generates more singlet oxygen than 8-MOP. Conveniently,oxygen and other gases may be removed from the inactivation medium bymaintaining the medium in a non-oxidizing gas atmosphere, e.g.,hydrogen, nitrogen, argon, helium, neon, carbon dioxide, and the like.The inactivation medium may be held in an enclosed vessel, and the spaceabove the liquid medium surface filled with the non-oxidizing gas.Oxidizing species initially in the medium will be exchanged for thenon-oxidizing gases according to gas-liquid equilibrium principles.Preferably, the space above the inactivation medium will be flushed withnon-oxidizing gas to remove the oxidizing species and further lowertheir equilibrium concentration in the liquid medium. Depending on thevolume of the inactivation medium, the flushing should be continued forat least 1 minute, preferably at least 2 minutes, usually being in therange from about 3 to 30 minutes. Flushing may be continued during theirradiation period, but need not be so long as the oxidizing specieshave been substantially removed and the vessel remains sealed to preventthe intrusion of air. Optionally, a singlet oxygen scavenger may beadded to the inactivation medium prior to irradiation to further preventinteraction of oxygen with the furocoumarin and the virus. Suitableoxygen scavengers include ascorbic acid, dithioerythritol, sodiumthionate, glutathione, and the like. The scavenger will be present at aconcentration sufficient to block active oxygen species, usually beingbetween 0.001M and 0.5M, more usually being between about 0.005M and0.02M, where the addition may be in single, multiple, or continuousadditions.

The concentration of dissolved oxygen may be reduced through the use ofenzyme systems, either in solution or immobilized on a solid substrate.Suitable enzyme systems include glucose oxidase or catalase in thepresence of glucose and ascorbic acid oxidose in the presence ofascorbate. Such enzyme systems may be employed alone or together withthe other methods for oxygen reduction discussed above.

The total time for the irradiation will vary depending upon the lightintensity, the concentration of the furocoumarin, the concentration ofthe virus, and the manner of irradiation of the virus, where theintensity of the irradiation may vary in the medium. The time ofirradiation necessary for inactivation will be inversely proportional tothe light intensity. The total time will usually be at least about 2hrs. and not more than about 60 hrs., generally ranging from about 10hrs. to 50 hrs. The times between additions of furocoumarin, where thefurocoumarin is added incrementally, will generally vary from about 1hour to 24 hrs., more usually from about 2 hrs. to 20 hrs.

The light which is employed will generally have a wavelength in therange from about 300 nm to 400 nm. Usually, an ultraviolet light sourcewill be employed together with a filter for removing UVB light. Theintensity will generally range from about 0. 1mW/cm² to about 5W/cm²,although in some cases, it may be much higher.

The temperature for the irradiation is preferably under 25° C., morepreferably under 20° C. and will generally range from about -10° C. to15° C., more usually from about 0° to 10° C.

During irradiation, the medium may be maintained still, stirred orcirculated and may be either continuously irradiated or be subject toalternating periods of irradiation and non-irradiation. The circulationmay be in a closed loop system or in a single pass system ensuring thatall of the sample has been exposed to irradiation.

It may be desirable to remove the unexpended furocoumarin and/or itsphotobreakdown products from the irradiation mixture. This can bereadily accomplished by one of several standard laboratory proceduressuch as dialysis across an appropriately sized membrane or through anappropriately sized hollow fiber system after completion of theirradiation. Alternatively, one could use affinity methods for one ormore of the low molecular weight materials to be removed.

The inactivated virus may then be formulated in a variety of ways foruse as a vaccine. The concentration of the virus will generally be fromabout 10⁶ to 10⁹ pfu/ml, as determined prior to inactivation, with atotal dosage of at least 10⁵ pfu/dose, usually at least 10⁶ pfu/dose,preferably at least 10⁷ pfu/dose. The total dosage will usually be at ornear about 10⁹ pfu/dose, more usually being about 10⁸ pfu/dose. Thevaccine may include cells or may be cell-free. It may be an inertphysiologically acceptable medium, such as ionized water,phosphate-buffered saline, saline, or the like, or may be administeredin combination with a physiologically acceptable immunologic adjuvant,including but not limited to mineral oils, vegetable oils, mineral saltsand immunopotentiators, such as muramyl dipeptide. The vaccine may beadministered subcutaneously, intramuscularly, intraperitoneally, orally,or nasally. Usually, a specific dosage at a specific site will rangefrom about 0.1 ml to 4 ml, where the total dosage will range from about0.5 ml to 8 ml. The number of injections and their temporal spacing maybe highly variable, but usually 1 to 3 injections at 1, 2 or 3 weekintervals are effective.

The following examples are offered by way of illustration, not by way oflimitation.

EXPERIMENTAL Materials and Methods A. Virus Growth and Tissue Culture

Hamster cells [BHK-21(C-13), American Type Culture Collection (ATCC),CCL 10]were grown as monolayers in plastic cell culture vessels inEagle's Minimum Essential Medium (MEM) with Earle's salts andnon-essential amino acids (MEN) supplemented with 10% heat inactivatedcalf serum (C^(i)) and 10% tryptose phosphate broth (Tp, e.g., Difco0060). Cell cultures were used to produce live vesicular stomatitisvirus, New Jersey serotype (VSV-NJ) from master seed virus originallyobtained from the ATCC (VR-159), and live bluetongue virus (BTV) frommaster seed virus originally obtained from Dr. T. L. Barber, USDA,Denver, Colorado. Cells were grown in culture vessels to 80% to 100%confluency (approximately 2×10⁵ cells per cm² of growth surface area)using standard mammalian cell culture techniques. Corning plastic rollerbottles (Corning No. 25140`-850) with a growth surface area of 850 cm²containing 100 ml of MEN supplemented with 10% C^(i) and 10% Tp and1×10⁸ to 2×10⁸ CCL 10 cells/bottle were used for virus production. Thecell cultures were initiated by seeding approximately 1×10⁶ to 5×10⁷cells into 100 mls of growth medium in a roller bottle containing 5% CO₂in air on a roller bottle rotator at 1 to 5 rpm at 35° C. to 38° C. Thecultures were grown to 80% to 100% confluency over a six to fourteen dayperiod with a medium change every two to four days.

When the monolayers reached 80% to 100% confluency, the culture mediumwas removed and the monolayer was infected with approximately 1×10⁶ to2×10⁶ plaque forming units (pfu) of VSV or BTV in 20 mls of MEN, with 2%heat-inactivated fetal bovine serum (F^(i)) added for BTV. Themultiplicity of infection (MOI) was approximately 0.01. The MOI mayrange from 0.001 pfu/cell to 0.033 pfu/cell. The virus inoculum wasadsorbed to the cells by incubation at 35° C. to 38° C. for one hour at1 to 5 rpm. One hundred mls of MEN containing 10% YELP supplement (v/v)for VSV, or 10% C^(i) and 10% Tp for BTV, was added per roller bottle.YELP supplement contains: yeast extract BBL 11929, 5 g/liter;lactalbumin hydrolysate GIBCO 670-1800, 25 g/liter; and Bacto-Peptone(Difco 0118), 50 g/liter. The post-infection incubation was carried outat 35° C. to 38° C. in 5% CO₂ /95% air with rotation. Sixteen toforty-eight hours post-infection, VSV cytopathic effect (CPE) wasevident, while BTV CPE became apparent from 2 to 4 days post infection.

The CPE was characterized by cell rounding, cell detachment, and celldegeneration. When visual or microscopic examination indicated that atleast 50% of the cell monolayer exhibited CPE, the contents of theroller bottle were swirled to remove loosely attached materials from theroller bottle walls. The harvest material was decanted from the rollerbottles into centrifuge bottles. The crude VSV harvest was clarified bycentrifugation at 500 to 1000×g for 20 minutes, at 4° C. The BTV harvestwas centrifuged at 2,000×g for 60 minutes at 4° C.

The clarified VSV preparations were concentrated by ultrafiltrationusing a Pellicon cassette system (Millipore XX42ASY60) with a cassettehaving a nominal exclusion limit of 10⁵ daltons (Millipore PTHK 000C5).The Pellicon cassette system was sterilized by filling the assembledunit with 1N NaOH and incubating the unit 12 to 24 hours at roomtemperature. The NaOH solution was pumped out of the cassette system andthe system was flushed with two to four liters of sterile H₂ O. Theassembly and operation of the Pellicon system were in accordance withthe instructions furnished by the manufacturer. All steps in theconcentration were performed aseptically. The clarified VSV wasconcentrated 15 to 40 fold, dimethylsulfoxide (Sigma D-5879) added to afinal concentration of 7.5% v/v, and suitable aliquots of the virusstored frozen at -80° C. to -100° C.

For BTV, the pellet resulting from centrifugation was resuspendedaseptically in 8 ml of 2mM Tris-HCl, pH 8.8, for each original rollerbottle. The suspension was mixed vigorously on a vortex mixer, and/orsonicated at 4° C. for 1 min., and centrifuged at 1,400×g for 30 min. at4° C. The virus-containing supernatant was collected and the pellet wasextracted twice more with 8 ml/roller bottle aliquots of 2mM Tris-HCl,pH 8.8. The virus-containing supernatants were pooled and clarified bycentrifugation at 4,000×g for 30 min. at 4° C. The clarified supernatantwas stored at 4° C.

Feline herpes I virus (FVR, the infective agent of feline viralrhinotracheitis) was grown as follows.

Cat cell lines AKD (ATCC CCL150) or Fc3Tg (ATCC CCL176) were grown asmonolayers in plastic cell culture vessels in a standard defined culturemedium consisting of MEN; F12K; MEM; or alpha MEM. Medium wassupplemented with 2% to 15% inactivated fetal calf serum (F^(i)) or 2%to 20% YELP. Cell cultures were used to produce live Feline Herpes Ivirus from master seed virus derived from Feline Herpes I virus (ATCCVR636). Cells were grown in culture vessels to 80% to 100% confluency(approximately 1×10⁵ to 2×10⁵ cells per cm² of growth surface area)using standard mammalian cell culture techniques as follows.

Corning plastic roller bottles containing 50 to 100 ml of MENsupplemented with 10% F^(i) and 1×10⁸ to 2×10⁸ AKD or Fc3Tg cells/bottlewere used for Feline Herpes I virus production. The cell cultures wereinitiated by seeding approximately 1×10⁶ to 5×10⁶ cells into 50 to 100mls of growth medium in a roller bottle containing about 5% CO₂ in airand incubating the roller bottle on a roller bottle rotator at 1 to 5rpm at 35° C. to 38° C. The cultures were grown to 80% to 100%confluency over a 7 to 14 day period with a 100% medium change every 3to 5 days.

When the monolayers were 80% to 100% confluent, the culture medium wasremoved and the monolayer was washed with 20 to 50 mls of phosphatebuffered saline (PBS) pH 7.2 to 7.4 (NaCl 8 g+KCl 0.2 g+Na₂ NPO₄ 1.14g+KH₂ PO₄ 0.2 g). The PBS wash was discarded, and the roller bottle wasinfected by the addition of approximately 1×10⁷ to 2×10⁷ plaque formingunits (pfu) of Feline Herpes I virus in 10 mls of PBS containing 2%F^(i). The multiplicity of infection (MOI) was approximately 0.1. Thevirus inoculum was adsorbed to the cells by incubation at 35° C. to 38°C. for one hour at 1 to 5 rpm. The inoculation fluid was removed and 50mls of MEN containing 10% F^(i) was added per roller bottle. Thepost-infection incubation was at 35° C. to 38° C. in 5% CO₂ in air withrotation. Herpesvirus cytopathic effect (CPE) was evident forty toforty-eight hours post-infection. The CPE was characterized by cellrounding, cell detachment, and cell degeneration.

The contents of the roller bottle were swirled 48 hours post-infectionto remove loosely attached materials from the roller bottle walls, andthe contents of the roller bottles were decanted into centrifugebottles. The virus, cells, and cell debris were pelleted bycentrifugation at 10,000×g for 30 minutes.

Cell associated (CA) Feline Herpes I virus was prepared by:

1. resuspending the 10,000×g pellet in approximately 5 ml of aresuspension medium containing 80 parts F12K, 10 parts F^(i), and 10parts dimethylsulfoxide (DMSO) for each original roller bottle;

2. freezing the resuspended CA virus at -20° C. for 1.5 to 2 hours; and

3. transferring the CA virus frozen at -20° C. to temperatures rangingfrom -80° C. to -100° C.

Cell free (CF) Feline Herpes I virus was prepared by:

1. resuspending the 10,000×g pellet in F12K;

2. freezing and thawing the resuspended material 3 times;

3. clarifying the freeze-thawed material by centrifugation at 10,000×gfor 30 minutes; and

4. freezing the clarified supernatant (CF virus) at temperatures rangingfrom -80° C. to -100° C.

CF or CA virus was thawed by gentle agitation at 37° C. in a water bath.

B. Virus Assay

Confluent monolayers of LMTK⁻, Vero (ATCC CCL 81), Fc3Tg, or AKD cellswere prepared in 6 cm diameter mammalian cell culture plastic petridishes (Corning #25010) or other convenient cell culture vessels. Thegrowth medium used for LMTK⁻ cells was alpha ME (alpha modified EaglesMinimum Essential Medium, Earle's Salts)+10% F^(i). The growth mediumused for Vero cells was MEN+5% F^(i). The growth medium used for Fc3Tgcells was MEN+10% F^(i), and the growth medium used for AKD cells wasF12K+15% F^(i) (VSV and BTV were titered on LMTK⁻ or Vero cells. FelineHerpes I was titered on Fc3Tg or AKD cells). Ten fold serial dilutionsof virus samples were made by adding 0.5 ml of the virus sample to 4.5mls of diluent (phosphate buffered saline, pH 7.2 to 7.4, plus 2% F^(i))in a screw cap tube. The growth medium was removed from a 6 cm culturedish cell monolayer, 0.1 ml of virus sample (undiluted or diluted) wasadded, and the virus was adsorbed to the monolayer for 1 to 2 hours at35° C. to 38° C. Two or more monolayers were used for each sample.

Five ml of overlay medium was added per 6 cm culture dish, except forFeline Herpes I, where the unadsorbed inoculum was removed, and 4 mls ofoverlay medium was added per 6 cm culture dish. The overlay medium forBTV or VSV was prepared by mixing equal parts of solution A (100 ml 2XMEM with L-glutamine, GIBCO #320-1935, +10 ml F^(i)) and 1.8% to 2%Noble Agar (Difco 0142) in deionized H₂ O at 44° C. to 45° C. Theoverlay medium for Feline Herpes I was prepared by mixing equal partssolution A and 1% methyl cellulose (4,000 centriposes) in deionized H₂ O(Fisher M-281 sterilized by autoclaving).

The virus infected cultures were incubated at 35° C. to 38° C. in 5% CO₂in air. Twenty-four hours before plaques were counted, a second overlaycontaining Neutral Red at a final concentration of 0.005% was added.Plaques were counted on day 2 or day 3 post-infection for VSV, on day 2to 4 for FVR and on day 6 or 7 for BTV. The virus titer in pfu/ml wascalculated by multiplying the average number of plaques per dish by thereciprocal of the dilution. The pfu/ml was the value used to determinethe amount of virus needed to infect cells at a MOI of approximately0.01. The pfu/ml in a virus preparation prior to inactivation was usedto determine the immunizing dose.

C. Virus Inactivation 1. VSV Inactivation

The thawed stock of VSV was pipetted into sterile T-150 tissue cultureflasks (nominally 25 ml into each of four flasks). To each flask wasadded 0.25 ml of 4'-aminomethyl-4,5', 8-trimethylpsoralen (AMT) stocksolution (stock solution is 1 mg/ml AMT dissolved in sterile, deionizedwater). Each flask was allowed to equilibrate in an argon atmosphere forat least 10 minutes. After equilibration, a stream of argon gas wasdirected into each flask for at least two minutes. The flasks were thentightly capped and placed under a long wavelength ultraviolet (320 nm to400 nm) light source (GE BLB fluorescent bulbs) at a temperature between0° C. and 20° C. for approximately 11 hours. The incident lightintensity was approximately 1mW/cm² (measured by a J-221 long wavelengthUV meter).

After the irradiation was completed, the flasks were removed from thelight source and an additional 0.25 ml of AMT stock solution was mixedinto each flask. The contents of each flask were pipetted into new,sterile T-150 flasks, and the flasks were again flushed with argon andirradiated for an additional 11 hours. This procedure was repeated threemore times until five additions (a total of approx. 50 μg/ml) of AMT hadbeen performed, the virus sample had been irradiated for at least 55hours, and at least four flask changes had been performed.

After all of the irradiations had been completed, the contents of theflasks were aseptically transferred to a common sterile container andstored at -85° C.

2. BTV Inactivation

Twenty-five ml of BTV serotype 11 (1.5×10⁸ pfu/ml) was mixed with 0.25ml of 4'-aminomethyl 4,5', 8-trimethylpsoralen (AMT; 1 mg/ml in DMSO).The mixture was placed in a 150cm² tissue culture flask (T-150; Corning#25120). The viral suspension in the flask was placed in an argonatmosphere for 10 min., and a stream of argon gas was then blown overthe viral suspension for an additional 2 min. The flask was tightlycapped and the suspension irradiated for 3.25 hrs. at 4° C. using GE BLBfluorescent bulbs at an intensity of 1.5mW/cm². An additional 0.25 ml ofAMT was then added to the viral suspension, the suspension transferredby pipette to a new T-150 flask, and the solution again flushed withargon. The flask was irradiated for an additional 14.75 hours at 4° C.under the same long wavelength UV light source. After this irradiationan additional 0.25 ml of AMT solution was added to the suspension, andit was again transferred to a new T-150 flask. The solution was flushedwith argon as before and irradiated for an additional 5.5 hrs. at 4° C.The inactivated BTV was stored at 4° C.

3. Feline Herpes I Inactivation a. Cell Free Virus

Nineteen mls of CF-FVR (1.9×10⁷ pfu/ml) were mixed with 0.4 ml ofhydroxymethyltrioxsalen (HMT; 1 mg/ml in DMSO) and 1.9 ml of sodiumascorbate (0.1 M in H₂ O). The mixture was prepared in 150 cm² tissueculture flasks (T-150, Corning No. 25120) that were subsequentlydeaerated for 2 minutes with pure argon gas. The virus-containing flaskswere irradiated for 55 minutes at 4° C. using G.E. BLB fluorescent bulbsat an intensity of 1.5 mW/cm². The FVR/HMT/ascorbate mixture was thentransferred by pipet into a second T-150 flask, which was deaerated for2 minutes using pure argon gas. The second T-150 flask was irradiatedfor an additional 28 minutes at 4° C. under the same long wavelength UVlight source.

The CF-FVR preparation was stored at -100° C. in a REVCO freezer.Subsequently the CF-FVR preparation was thawed and placed into a T-150flask. The flask was deaerated with pure argon gas for 2 minutes andirradiation was continued as described above for an additional 15 hoursand 40 minutes.

b. Cell Associated Virus

Cells from 10 roller bottles (about 1×10⁸ to 2×10⁸ cells/roller bottle)were resuspended in 28 mls of cell culture media. Twenty mls of thesuspension were placed into a T-150 flask. To this flask was added 2 mlof freshly prepared sterile 0.1 M sodium ascorbate and 0.4 ml HMT (1mg/ml in DMSO). The flask was deaerated with pure argon gas for 2minutes, and the flask was irradiated at 4° C. using G.E. BLBfluorescent bulbs at an intensity of 1.5 mW/cm² for 75 minutes. Theviral suspension was then transferred by pipet from the T-150 flask intoa second T-150 flask and again deaerated with pure argon gas for 2minutes. Irradiation was continued for an additional 95 minutes. TheCA-FVR preparation was adjusted to 10% DMSO and the suspension wasfrozen at -20° C. for 1 hour and then stored at -100° C. in a REVCOfreezer.

The stored frozen CA-FVR preparation was subsequently thawed, and thecells were pelleted in a clinical centrifuge. The cells were resuspendedin 21 mls of serum-free medium to which 2.1 mls of freshly prepared 0.1M sodium ascorbate and 0.4 ml of HMT (1 mg/ml in DMSO) were added. Thesample was transferred by pipet to a T-150 flask, and irradiation wascontinued for an additional 15 hours and 40 minutes.

Results A. Bluetongue Virus 1. Assessment of Inactivation by BlindPassage

CCL 10 cells were grown to confluency in 850 cm² roller bottles usingstandard cell culture procedures, as described above. The culture mediumwas removed from the roller bottle, and 2.0 ml of the inactivated viruspreparation mixed with 18 ml of medium containing 2% F^(i) was adsorbedto the roller bottle cell monolayer for 60 min at 35° C. to 38° C. withrotation at 1 to 5rpm. After adsorption, the residual unabsorbedinoculum was removed, and 100 ml of growth medium (MEN with 10% C^(i)and 10% Tp) was added and the roller bottle culture incubated at 35° C.to 38° C. for 7 days with daily observation for viral CPE. The rollerbottle culture received a 100% medium change every 2 to 3 days. If noCPE was observed during the first roller bottle passage, the cellmonolayer was chilled at 4° C. for 12 to 24 hrs. The cells were scrapedinto the medium which was then decanted into a centrifuge bottle. Thecells were pelleted by centrifugation at 4° C. at 2,000×g for 30 min.and resuspended in 2.0 ml of 2mM Tris-HCl (pH 8.8) by vigorous mixingusing a vortex mixer. The resuspended material was centrifuged at2,000×g for 20 min. at 4° C. The supernatant was added to 18 ml ofgrowth medium containing 2% F^(i) and used to infect a new confluentroller bottle culture of CCL 10 cells, as described immediately above.The second roller bottle blind passage was observed for 7 days and fedevery 2 to 3 days. If no CPE was observed during the second rollerbottle blind passage, a third roller bottle blind passage was performed.If no CPE had been observed by the end of the third roller bottle blindpassage the virus preparation was considered inactivated and suitablefor in vivo testing.

2. Immunization of Rabbits with Psoralen-inactivated BTV Vaccine a.Example 1

Four New Zealand white rabbits were randomly assigned to 2 groups,designated A and B. Both groups were given 4 immunizations at two weekintervals. The first immunization consisted of 1 ml of vaccine (10⁸ pfuBTV serotype 11) and 1 ml of Freund's Complete Adjuvant. The secondthrough fourth immunizations utilized 1 ml of vaccine (10⁸ pfu BTVserotype 11) and 1 ml of Freund's Incomplete Adjuvant. All immunizationswere given intramuscularly (IM). The vaccine given to Group A (Vaccine#1) was inactivated with AMT-UVA in the presence of 0.01M ascorbic acid.Vaccine #1 was dialyzed for 12 hours against 2mM Tris, pH 8.6. Thevaccine given to Group B (Vaccine #2) was inactivated with AMT-UVAwithout ascorbic acid and sonicated three times (2 minutes each time)using a cup horn probe (Heat Systems Model 431A) at a power setting of 3(Heat Systems Model W220). Both Vaccine # 1 and Vaccine #2 were deemedinactivated since no live virus was detected during blind passage.Inactivated vaccine was also tested for safety by chicken embryoinoculation. Egg deaths attributable to live virus were not encountered.Both rabbit groups were bled via auricular venipuncture one weekfollowing the second, third, and fourth immunizations. Serum from eachrabbit was pooled with that of its groupmate, and the pooled sera weretested for anti-BTV antibodies by two standard serologic assays, serumneutralization (Jochim and Jones, Am. J. Vet. Res. (1976) 37:1345-1347)and agar gel precipitation (Jochim et al., Am. Assoc. Vet. Lab. Diag.,22nd Proceed.: 463-471, 1979) Pre-immunization rabbit serum was used asthe negative control; BTV immune sheep serum was used as the positivecontrol for both immunologic procedures.

Pooled sera from Groups A and B reduced the number of viral plaques(serum neutralization) greater than eighty percent (arbitrarily selectedend point) when the sera were diluted 1:40, which was the highestdilution examined Negative and positive control sera behaved asexpected.

                  TABLE 1                                                         ______________________________________                                        Serum Neutralization Data From Rabbits                                        Vaccinated with AMT-UVA-inactivated                                           Bluetongue Virus Vaccines.                                                                    Titer*:                                                       Group             1          5     40                                         ______________________________________                                        A                 +          +     +                                          B                 +          +     +                                          Normal Rabbit Serum                                                                             -          -     -                                          BTV-Immune Sheep Serum                                                                          +          +     ±                                       ______________________________________                                         *Reciprocal of serum dilution neutralizing 80 percent of BTV plaque           activity on BHK cells. The data are from the postsecond immunization seru     samples.                                                                 

Pooled post-immunization sera from Groups A and B precipitated BTVantigen in immunodiffusion plates when tested at dilutions up to 1:16.Normal rabbit serum did not precipitate the standard BTV antigen.BTV-immune sheep serum did precipitate the BTV antigen, but not atdilutions greater than 1:2.

Of the two immunologic procedures utilized, serum neutralization isconsidered predictive for immunity to live BTV challenge in the targetspecies.

b. Example 2

Twelve New Zealand white rabbits were randomly assigned to six groups,A-F, two rabbits per group. An additional four rabbits were assigned toGroup G. These sixteen rabbits were vaccinated twice subcutaneously withthe AMT-UVA inactivated Bluetongue virus vaccines described in Table 2.Preinactivation titer was approximately 10⁸ pfu for each serotype. Thevaccines were formulated with 20% (v/v) aluminum hydroxide adjuvant, andwere given with a three week interval between the first and secondinoculations.

The sixteen rabbits were bled by auricular venipuncture on days 0, 14and 35. Each serum was heat-inactivated and tested against BTV serotypes10, 11, 13 and 17 for serum neutralizing antibody. All vaccinatedrabbits developed SN titers against the homologous vaccine serotypes(Table 3). These data demonstrated the immunopotency of a multivalentAMT-UVA inactivated Bluetongue virus vaccine.

                  TABLE 2                                                         ______________________________________                                        Serotype Composition of Inactivated Bluetongue                                Virus Vaccines Tested in Rabbits                                                                        BTV Serotype                                        Group       Rabbit #      Composition                                         ______________________________________                                        A            1, 2         10                                                  B            3, 4         11                                                  C            5, 6         13                                                  D            7, 8         17                                                  E            9, 10        11, 17                                              F           11, 12        10, 11, 17                                          G           13, 14, 15, 16                                                                              10, 11, 13, 17                                      ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Serum Neutralizing Data from Rabbits Vaccinated                               with AMT-UVA Single and Multi-Serotype Bluetongue                             Virus Vaccines                                                                         SN Titer* Against:                                                   Group  Rabbit  BTV-10   BTV-11  BTV-13 BTV-17                                 ______________________________________                                        A       1      1:160    1:10    1:10   1:10                                           2      1:320    1:10    1:10   1:10                                   B       3      1:10     1:320   1:10   1:10                                           4      1:10     1:80    1:10   1:10                                   C       5      1:20     1:20    1:160  1:10                                           6      1:20     1:10    1:40   1:10                                   D       7      1:10     1:10    1:10   1:320                                          8      1:10     1:10    1:10   1:320                                  E       9      1:20     1:160   1:20   1:160                                         10      1:20     1:160   1:20   1:160                                  F      11      1:160    1:160   1:20   1:160                                         12      1:40     1:40    1:20   1:80                                   G      13      1:160    1:160   1:80   1:160                                         14      1:160    1:160   1:80   1:160                                         15      1:160    1:160   1:40   1:160                                         16      1:80     1:160   1:160  1:160                                  ______________________________________                                         *Reciprocal of serum dilution neutralizing 80% of BTV plaque activity on      Vero cells. The data are from the postsecond immunization sera (Day 35).      Negative and positive control sera behaved as expected in the SN assay.  

3. Immunization of Sheep with Psoralen-inactivated BTV Vaccine a.Example 1

Each of two adult sheep, known to be susceptible to BTV, was inoculatedsubcutaneously (SQ) with 2 ml of AMT-UVA inactivated BTV plus adjuvant(1:1; vaccine to aluminum hydroxide adjuvant). The vaccine containedapproximately 10⁸ pfu/ml of BTV prior to inactivation. A third sheep wasinoculated SQ with 6 ml of the identical vaccine without adjuvant. Sevenweeks later the three sheep were given identical inoculations SQ thatconsisted of 5 ml of vaccine and aluminum hydroxide adjuvant (2:1vaccine to adjuvant; 10⁸ pfu BTV/ml of vaccine).

The three sheep were monitored for clinical evidence of BTV, includingdaily body temperature recording and bi-daily virus isolation attempts.No evidence of BTV was observed, indicating that the vaccine wasinactivated.

Serum was collected weekly for serum neutralization and agar gelprecipitation testing. Normal sheep sera and BTV-immune sheep sera wereused for negative and positive control samples in the serologic tests.

The first vaccine inoculations induced precipitating anti-BTV antibodyin all three sheep. Their pre-exposure sera were uniformly negative foranti-BTV precipitating antibody. Modest neutralizing anti-BTV antibodytiters (1:5) were elicited in two of three sheep following oneimmunization. The second immunization elicited a distinct immunologicanamnestic response, inducing neutralizing titers of 1:40, 1:80, or1:160 in the three sheep.

                  TABLE 4                                                         ______________________________________                                        Serum Neutralization Data From Sheep Immunized                                with an AMT-UVA Inactivated BTV Vaccine.                                                     TITERS*                                                                       Sheep No.:                                                                    1       2      3                                               ______________________________________                                        Pre-First Immunization                                                        Day 0            <5        <5     <5                                          Post-First Immunization                                                       Day 21            5         5     <5                                          Post-Second Immunization                                                      Day 7            80        160    40                                          Day 14           80         40    40                                          Day 21           80         80    40                                          Day 42           80         80    80                                          Post-Challenge                                                                Day 7            160       160    80                                          Day 14           320       160    80                                          ______________________________________                                         *Reciprocal of highest 2fold dilution reducing BTV plaque activity on BHK     cells by 80 percent.                                                     

The sheep were challanged by SQ syringe inoculation of 10⁵ egg lethaldoses of BTV serotype 11. The three sheep remained clinically normalduring the BTV challenge period, indicating that the vaccine wasefficaceous.

It is evident from the above results that the BTV which ispsoralen-inactivated retains its immunogenicity, particularly as tothose sites which elecit an immune response which is effective inprotecting a host against subsequent BTV-infection. Thus, the psoraleninactivation can be carried out under conditions which do not modify theimmunogenic sites of the virus, so as to elecit an immunogenic responsewhich will be effective against the live BTV. Furthermore, the BTV RNAvirus is efficiently inactivated under mild conditions to the point ofcomplete inactivation, whence it may be safely administered to a host.

b. Example 2

Eight experimental and four control sheep, known to be Bluetongue Virussusceptible, were housed together in an insect-proof facility. Theexperimental sheep were inoculated twice subcutaneously with AMT-UVAinactivated BTV Serotype 11 vaccine. Each vaccinate receivedapproximately 3×10⁸ pfu BTV-11 formulated with twenty-five percent (v/v)aluminum hydroxide adjuvant. Three weeks elapsed between immunizations.Control sheep were inoculated with tissue culture fluid in 25% percent(v/v) aluminum hydroxide. Serum samples were collected prior tovaccination, following vaccinations, and following challenge, and testedfor SN antibodies. All sheep were challenged by subcutaneous inoculationof 2×10⁵ ELD₅₀ BTV-11 four weeks post-second vaccination. Virusisolation was performed twice weekly post-challenge for six weeks. Virusisolation from sheep blood was done by intravenous chicken embryoinoculation, followed by specific BTV serotype identification byneutralization in vitro.

Five of the eight vaccinated sheep developed SN titers of 1:20post-second vaccination. All eight vaccinates resisted subcutaneouschallenge with 2×10⁵ ELD₅₀ BTV-11, whereas the four control sheepdeveloped uniform viremia as assessed by egg inoculation. Sheep data aregiven in Table 5.

                  TABLE 5                                                         ______________________________________                                        Serum Neutralization and Virus Isolation Data from Sheep                      Vaccinated with AMT-UVA Inactivated BTV-11 Vaccine and                        Subsequently Challenged with 2 × 10.sup.5 ELD.sub.50 of Live            BTV-11                                                                                      SN Titer           Virus Isolation                              Sheep Base-   Post-Second                                                                              Post-   Post-Challenge Day                           No.   line    Vaccination                                                                              Challenge                                                                             4    11  15  18                              ______________________________________                                        Exper-                                                                        imen-                                                                         tal                                                                           650   neg     1:20       1:160   -    -   -   -                               651   neg     1:20       1:40    -    -   -   -                               652   neg     1:20       1:160   -    -   -   -                               653   neg     1:20       1:40    -    -   -   -                               656   neg     1:10       1:160   -    -   -   -                               658   neg     1:10       1:40    -    -   -   -                               659   neg     1:20       1:160   -    -   -   -                               660   neg     1:10       1:160   -    -   -   -                               Controls                                                                      654   neg     neg        1:10    +    +   +   +                               655   neg     neg        neg     +    +   +   +                               661   neg     neg        1:40    +    +   +   +                               662   neg     neg        1:160   +    +   +   -                               ______________________________________                                    

B. Feline Herpes Virus I 1. Assessment of Inactivation by Blind Passage

Fc3Tg or AKD cells were grown to confluency in 850 cm² roller bottlesusing standard cell culture procedures as described above. The culturemedium was removed from the roller bottle, and 2.0 mls of theinactivated virus preparation, mixed with 18 mls of medium containing 2%F^(i), were adsorbed to the roller bottle cell monolayer for 60 minutesat 35° C. to 38° C. with rotation at 1 to 5 rpm. After adsorption, theinoculum was removed and 150 ml of maintenance medium (MEN or F12K with2% F^(i)) added. The roller bottle culture was then incubated at 35° C.to 38° C. for 7 days with daily observation for viral CPE. The rollerbottle culture received a 100% medium change after 3 to 5 days. If noCPE was observed during the first roller bottle passage, the cellmonolayer was scraped into the maintenance medium which was thendecanted into a centrifuge bottle. The cells were pelleted bycentrifugation at room temperature at 1,000 x g for 15 minutes,resuspended in 20 ml of fresh maintenance medium, and passed to a newconfluent roller bottle culture of Fc3Tg or AKD cells as describedabove. The second roller bottle blind passage was observed for 7 daysand fed once on day 3 to 5. If no CPE was observed during the secondroller bottle blind passage, a third roller bottle blind passage wasperformed. If no CPE was observed by the end of the third roller bottlepassage, the virus preparation was considered inactive.

2. Administration Procedure for Psoralen-inactivated FVR Vaccines

Photochemically inactivated FVR was inoculated via syringe into cats byvarious routes, including but not limited to intravenously (IV),subcutaneously (SQ), intramuscularly (IM), or intraperitoneally (IP).The vaccine was administered in various volumes (0.5 to 3.0 ml) and invarious concentrations (10⁶ to 10⁸ pfu; either CF, CA or incombination). In the following examples, the vaccine was administered incombination with aluminum hydroxide as an immunologic adjuvant. Thenumber of injections and their temporal spacing was as set forth in eachexample.

3. Immunization with Psoralen-inactivated CF-FVR Vaccine

The experimental group consisted of four specific pathogen free kittens(2 males, 2 females) four months old (Liberty Laboratories, LibertyCorner, N.J.). The control group consisted of two similar femalekittens. The experimental group was inoculated IM with 3×10⁷ pfu (3 mls)of HMT inactivated CF-FVR on days 0 and 21, and again inoculated with3×10⁷ pfu HMT inactivated with an equal amount of 2% aluminum hydroxide[Al(OH)₃ ] adjuvant on day 61. Controls were vaccinated at eight weeksand at thirteen weeks of age with a commercial FVR vaccine using themanufacturer's recommended procedure. Sireum samples were collectedweekly and tested for anti-FVR neutralizing antibodies.

Following live virus challenge (10⁶ pfu intranasally andintraconjunctivally), a numerical scoring system (Table 6) was used toassess the clinical response of both experimental and control cats.

                  TABLE 6                                                         ______________________________________                                        Scoring System for Clinical                                                   Effects of Herpesvirus Challenge in Cats                                      Factor         Degree        Score                                            ______________________________________                                        Fever          101 to 102° F.                                                                       0                                                               102 to 103    1                                                               103 to 104    3                                                               greater than 104                                                                            5                                                Depression     slight        1                                                               moderate      3                                                               severe        5                                                Sneezing       occasional    1                                                               moderate      3                                                               paroxysmal    5                                                Lacrimation    serous        1                                                               mucoid        3                                                               purulent      5                                                Nasal Discharge                                                                              serous        1                                                               mucoid        3                                                               purulent      5                                                Appetite       normal; eats all food                                                                       0                                                               fair; eats more than                                                                        1                                                               1/2 of food                                                                   poor; eats less than                                                                        3                                                               1/2 of food                                                                   none; eats nothing                                                                          5                                                ______________________________________                                    

Three of four experimental cats developed serum neutralizing anti-FVRantibody (SN) titers of 1:2 that were detected between day 42 and day58. Following the third immunization (day 61), four of four experimentalcats had SN titers of 1:4 (day 80). Baseline SN antibody titers on theexperimental cats were negative. The control cats did not developdetectable SN antibody titers during the pre-challenge period.

All cats were exposed to 10⁶ pfu of live FVR by intraconjunctival andintranasal exposure on day 91. Each cat was monitored twice daily forthe absence, presence and degree of severity of factors given in Table6. A composite clinical score was derived for each cat after a 15 dayobservation period.

Three of four experimental cats demonstrated mild temperature elevationand serous ocular or nasal discharge along with mild intermittentdepression and appetite suppression. Their composite scores were 39, 42,and 35 respectively for the 15 day observation period. The fourthexperimental cat was more severly affected (composite score =84) bymoderate, but transient, sneezing and mucoid nasal discharge. Bothcontrol cats were severely affected by live virus challenge. Severepurulent nasal and ocular discharge and lack of appetite were apparent.The control cats had composite scores of 133 and 253.

Three weeks following live FVR challenge, all cats were tested for SNantibody titers against FVR. Three of four experimental cats had SNantibody titers of 1:16 while the fourth cat had a 1:8 titer. One of thecontrol cats had an SN antibody titer of 1:4 while the second controllacked an SN antibody titer against FVR.

4. Immunization with Psoralen-inactivated CA-FVR Vaccine

Nine age-matched specific pathogen free kittens, 4 months old (LibertyLaboratories, Liberty Corner, N.J.), were randomly assigned to threeexperimental groups designated A, B, and C.

Group A (controls) was inoculated twice with 1 ml tissue culture fluidand 1 ml aluminum hydroxide adjuvant. Group B was inoculated twice witha commercial FVR vaccine according to the manufacturer's recommendation.Group C was inoculated three times with 10⁷ HMT-inactivated CA-FVR inaluminum hydroxide (total volume =2 ml; 1:1 vaccine to adjuvant). Allinjections were given IM at three week intervals.

Live FVR virus (10⁶ pfu intranasally and intraconjunctivally) was givenon day 63 and a numerical scoring system (Table 6) was used to assessthe kittens' clinical response for a 15 day post-challenge period. Serumsamples were collected from all kittens prior to vaccination, prior tothe second and third immunizations, prior to live FVR challenge, and at15 days post-challenge. The sera were utilized to assess neutralizingantibody titers by standard procedures.

The control kittens (Group A) maintained SN antibody titers less than1:2 (negative) throughout the pre-challenge period. Fifteen daysfollowing live FVR challenge Group A kittens uniformly had SN antibodytiters of 1:2. Kittens in Groups B and C lacked detectable anti-FVRantibody titers pre-immunization, but all kittens in Groups B and C hadSN antibody titers of 1:2 or 1:4 after two immunizations. The thirdimmunization in Group C kittens did not significantly alter their SNantibody titers. Following a 15 day post-challenge period, kittens inGroups B and C demonstrated an anamnestic immunologic response, with SNantibody titers ranging from 1:16 to 1:64.

Clinically, Group A kittens were severely affected by live FVRchallenge, whereas kittens in Groups B and C were significantlyprotected by their respective vaccines.

The composite clinical scores for Group A were 125, 141, and 128 for the15 day post-challenge period. The composite clinical scores for Group Bwere 25, 20, and 64, while Group C had composite clinical scores of 21,15, and 34. The clinical signs evident were characteristic of FVR.

From the SN data and clinical scoring, it is evident that kittensimmunized with the experimental HMT-inactivated FVR vaccines (cell-freeor cell associated) in the above examples were significantly immune tothe clinical effects of severe FVR challenge.

C. Vesicular Stomatitis Virus 1. Assessment of Inactivation byIntracerebral Inoculation of Mice

Suckling mice (0 to 10 days old) were inoculated intracerebrally with0.02 ml of the psoralen-inactivated VSV-NJ using a tuberculin syringeand a 28 or 30 gauge needle. Each vaccine lot was tested in four to ninesuckling mice. The mice were observed three times daily for a minimum ofseven days. Residual low-level live VSV kills suckling mice in two tofive days. The sensitivity of this assay is approximately 1 to 5 pfu oflive VSV per intracerebral dose. Inactivated VSV-NJ vaccine wasconsidered safe (inactivated) if all inoculated suckling mice survivedthe seven day observation period. The VSV-NJ vaccine batches usedhereinafter each passed the suckling mouse safety test prior to use.

2. Virus Neutralization in Mice Immunized with Psoralen-inactivatedVSV-NJ Vaccine

Groups of ten adult white mice each were injected using threeimmunological adjuvants (aluminum hydroxide gel, incomplete Freund's, oroil emulsion) with one of three psoralen-inactivated VSV-NJ vaccinedoses (10⁹, 10⁸, or 10⁷ pfu/dose). The oil emulsion was prepared asdescribed by Stone et al. (1978) Avian Dis. 22:666-674. All mice wereinjected IP once each, on day 0 and day 21. Serum samples were collectedfrom the orbital sinus on day 20 and on day 33 and pooled serum sampleswere assessed for serum neutralization (SN) activity by standardprocedures. See, Castaneda et al. (1964) Proc. US Livestock San. Assoc.68:455-468. Serum samples were negative for neutralizing antibodies toVSV-NJ prior to vaccination.

The vaccine with oil emulsion adjuvant induced the highest SN titersafter one injection. All three vaccine doses, regardless of adjuvant,induced SN titers of at least 1:2000 after two injections. Serumdilutions were tested for SN activity only to 1:2560. The results areset forth in Table 7.

                  TABLE 7                                                         ______________________________________                                        Virus Neutralization Indices* of Mouse Sera                                   After One and Two Injections of Psoralen-                                     Inactivated VSV-NJ Vaccine                                                                        Log.sub.10 of Vaccine Con-                                            No. of  centration (pfu/dose)                                     Adjuvant      Injections                                                                              7        8     9                                      ______________________________________                                        Aluminum hydroxide gel                                                                      1            67*     905   905                                  Aluminum hydroxide gel                                                                      2         >2560     2560 >2560                                  Freund's Incomplete                                                                         1           226      57    905                                  Freund's Incomplete                                                                         2          2033    >2560 >2560                                  Oil Emulsion  1         >2560    >2560  2357                                  Oil Emulsion  2         >2560    >2560 >2560                                  ______________________________________                                         *Virus neutralization index is the reciprocal of the serum dilution that      neutralized 32 TCID.sub.50 of VSVNJ.                                     

3. Virus Neutralization in Hamsters Vaccinated with Psoralen-inactivatedVSV-NJ Vaccine

Groups of five MHA hamsters each were injected with either 10⁹, 10⁸, or10⁷ pfu psoralen-inactivated VSV-NJ per dose, with or without aluminumhydroxide adjuvant (1:1). All hamsters were injected intramuscularly(IM) once each, on day 0 and again on day 21. Pooled serum samples werecollected on day 21 and on day 34 for serum neutralization testing bystandard procedures. Serum neutralizing antibodies were elicited by allthree vaccine doses tested, with or without aluminum hydroxide adjuvant.SN titers are given in Table 8.

                  TABLE 8                                                         ______________________________________                                        Virus Neutralization Indices* of Hamster                                      Sera After One and Two Injections of                                          Psoralen-Inactivated VSV-NJ Vaccine                                                               Log.sub.10 of Vaccine Con-                                             No. of centration (pfu/dose)                                     Adjuvant       Injections                                                                             7        8    9                                       ______________________________________                                        None           1         134*     134  1076                                   None           2        1280     1810 >2560                                   Aluminum hydroxide gel                                                                       1         538      538 >2560                                   Aluminum hydroxide gel                                                                       2        1810     1920  2560                                   ______________________________________                                         *Virus neutralization index is the reciprocal of the serum dilution that      neutralized 32 TCID.sub.50 of VSVNJ.                                     

4. Live VSV-NJ Challenge of Mice Vaccinated with Psoralen-inactivatedVSV-NJ Vaccine

Three groups of fourteen, sixteen and seventeen adult white mice eachwere injected with either 10⁷, 10⁶ or 10⁵ pfu psoralen-inactivatedVSV-NJ per dose, respectively, using oil emulsion adjuvant with allinjections. Each mouse was injected once IP (day 0). Pooled serumsamples were collected on day 0 and again on day 21, and these sampleswere tested for SN antibody titers by standard procedures. The resultsare set forth in Table 9.

                  TABLE 9                                                         ______________________________________                                        Virus Neutralization Indices* of Mouse                                        Sera After One Injection With Psoralen-                                       Inactivated VSV-NJ Vaccine, Using Oil                                         Emulsion Adjuvant                                                             Log.sub.10 of Vaccine Concentration                                           (pfu/dose)                                                                    Day     5             6        7                                              ______________________________________                                         0       --*          --       --                                             21      --            --       40                                             ______________________________________                                         *Virus neutralization index is the reciprocal of the serum dilution that      neutralized 56 TCID.sub.50 of VSVNJ                                      

Each group of mice was subdivided into three groups of about five miceeach. Each mouse group was challenged with either 1, 10 or 100 minimumlethal doses (MLD) of live VSV by intracerebral inoculation on day 33.

Two of five mice that were immunized with 10⁶ pfu psoralen-inactivatedVSV-NJ survived a one MLD VSV challenge but five of five mice that wereimmunized with 10⁷ pfu psoralen-inactivated VSV-NJ vaccine survived botha 1 or 10 MLD VSV challenge. One of four mice that were vaccinated at10⁷ pfu/dose psoralen-inactivated VSV-NJ survived a 100 MLD VSVchallenge. The results (no. dead/no. challenged) are set forth in Table10.

                  TABLE 10                                                        ______________________________________                                        Live VSV-NJ Challenge of Mice Injected with                                   Psoralen-Inactivated VSV-NJ                                                   Dose Psoralen-                                                                           Challenge Dilution                                                 Inactivated                                                                              10.sup.-5   10.sup.-4 10.sup.-3                                    VSV-NJ Vaccine                                                                           (1 MLD)     (10 MLD)  (100 MLD)                                    ______________________________________                                        10.sup.7 pfu                                                                              0/5*       0/5       3/4                                          10.sup.6 pfu                                                                             3/5         4/5       3/6                                          10.sup.5 pfu                                                                             5/5         4/5       7/7                                          ______________________________________                                         *Number dead/number challenged                                           

5. Virus Neutralization in Cattle Immunized with Psoralen-inactivatedVSV-NJ Vaccine

Four groups of six mature beef cattle each were injected with either 10⁸or 10⁷ pfu/dose psoralen-inactivated VSV-NJ vaccine, with or withoutaluminum hydroxide adjuvant (1:1). Each cow was vaccinatedsubcutaneously (SQ) on day 0 and again on day 21. A control groupconsisted of an additional six cattle that were inoculated only withadjuvant on day 0 and again on day 21. All cattle were bled on days 0,14, 21, and 35. Serum from each animal was tested for SN antibodies toVSV-NJ by standard procedures.

The aluminum hydroxide adjuvant was required to elicit significant SNtiters in cattle, and 10⁸ pfu/dose induced the highest responses. Theresults are set forth in Table 11. A VSV-NJ virus neutralization indexgreater than 1000 has been reported to represent protection against 10⁶ID₅₀ of live VSV by intralingual challenge in cattle. See, Castaneda etal. (1964) Proc. US Livestock San Assoc. 68:455-468.

                  TABLE 11                                                        ______________________________________                                        Virus Neutralization Indices* From Cattle                                     Injected With Psoralen-Inactivated VSV-NJ                                     Vaccine                                                                                       Day Serum Collected                                           Group  Treatment  Animal  0**  14   21**  35                                  ______________________________________                                        A      10.sup.8 pfu/                                                                            310     --   16   16     256                                       dose +     731     --   --   --    > 16                                       Al(OH).sub.3                                                                             911     --   128  64    2048                                                  921     --    8    8    1024                                                  943     --   16   32    1024                                                  944     --   32   32     512                                B      10.sup.7 pfu/                                                                            303     --   --   --     256                                       dose +     304     --   --   --     64                                        Al(OH).sub.3                                                                             308     4     4    8     512                                                  542     --   --   --      8                                                   914     --   16    4     512                                                  1670    --   --   --    >128                                C      Controls   305     --   --   --    --                                                    309     --   --   --    --                                                    314     --   --   --    --                                                    315     --   --   --    --                                                    316     --   --   --    --                                                    318     --   --   --    --                                  D      10.sup.8 pfu/                                                                            302     --   --   --      4                                        dose       611     --   --   --      4                                        without    714     --   --   --      8                                        adjuvant   732     --   --   --      4                                                   747     --   --   --    --                                                    996     --   --   --     32                                 E      10.sup.7 pfu/                                                                            101     --   --   --    --                                         dose       312     --   --   --      4                                        without    616     --   --   --    --                                         adjuvant   721     --   --   --    --                                                    722     --   --   --    --                                                    1944    --   --   --    --                                  ______________________________________                                         *Virus neutralization index is the reciprocal of the serum dilution that      neturalized 32 TCID.sub.50 of VSVNJ.                                          **Immunization Days                                                      

6. Live VSV-NJ Challenge of Cattle Vaccinated with Psoralen-inactivatedVSV-NJ Vaccine

Ten mature cattle were divided into two groups of five animals each.Group I was designated experimental and Group II was designated control.All ten cattle were clinically normal and lacked evidence of previousVSV exposure; that is, they were negative for serum neutralizing (SN)antibody. Group I cattle were vaccinated subcutaneously with 10⁸ pfu(prior to inactivation) psoralen-inactivated VSV twice with a three weekinterval. Vaccine volume was 2 ml, containing aluminum hydroxideadjuvant. Group II cattle were not exposed to the psoralen-inactivatedVSV.

Approximately two weeks post-second vaccination, the cattle of bothGroups I and II were challenged intradermalingually with 0.1 ml live VSVin log dilutions of 5.6 pfu to 5.6×10⁵ pfu/injection site. Thus eachanimal's tongue received six separate 0.1 ml injections, representing aquantitative challenge system. Serum neutralizing titers for cattle ineach group measured before and after challenge are presented in Table12.

                  TABLE 12                                                        ______________________________________                                        Serum Neutralization Titers From Cattle                                       Vaccinated With Psoralen-Inactivated VSV-NJ                                   Vaccine                                                                       Arri-     After    After     Day of  Post                                     val       1st vacc.sup.a                                                                         2nd vacc.sup.b                                                                          Challenge.sup.a                                                                       Challenge.sup.c                          Animal                                                                              Day                                                                     No.   0       18       35      42      60                                     ______________________________________                                        Group                                                                         I:                                                                            4009-V                                                                              neg*    1:160    1:1280  1:1280  1:1280                                 4383-V                                                                              neg     1:80     1:1280  1:1280  1:2560                                 4389-V                                                                              neg     1:80     1:640   1:2560  ND                                     6153-V                                                                              neg     1:80     1:1280  1:1280  ≧1:20480                        6246-V                                                                              neg     1:320    1:1280  1:1280  ND                                     Group                                                                         II:                                                                           3780-C                                                                              neg     neg      neg     neg     1:10240                                3781-C                                                                              neg     neg      neg     neg     1:10240                                3784-C                                                                              neg     neg      neg     neg     1:10240                                4007-C                                                                              neg     neg      neg     neg     1:10240                                7912-C                                                                              neg     neg      neg     neg     1:10240                                ______________________________________                                         .sup.a 100 TCID.sub.50 of VSVNJ                                               .sup.b >1000 TCID.sub.50 of VSVNJ                                             .sup.c 37 TCID.sub.50 of VSVNJ                                                *negative at 1:20, the lowest dilution tested                                 ND = not done                                                            

Vaccinated animals had a fifty percent reduction in lesion number, andlesions on vaccinates were fifty percent smaller and healed faster thanon controls. Control animals developed lesions at both earlier and latertime points. On post-challenge day eighteen, all five controls hadlesions whereas four of five vaccinates were normal. The fifthvaccinate's lesions were milder than those of any control animal onpost-challenge day eighteen.

Using the Mann-Whitney modification of Wilcoxon's two sample test, thevaccinates were significantly protected against live VSV challenge(P=.075). On the average, vaccinated cattle were protected against 25times the minimum infectious dose required to produce lesions in controlanimals.

According to the present invention, viruses inactivated withfurocoumarins and ultraviolet radiation in the substantial absence ofoxygen and other oxidizing species retain their immunogenicity and aresuitable as the immunogenic substance in vaccines against a number ofvirally-induced diseases. The inactivated viruses of the presentinvention are non-infectious and safe when administered to a host forvaccination, yet display enhanced antigenic integrity when compared tovaccines inactivated in the presence of oxygen.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A method for inactivating a live virus withoutsubstantially degrading their antigenic characteristics, said methodcomprising:exposing the virus to a preselected intensity of longwavelength ultraviolet radiation and a preselected concentration of aninactivating furocoumarin for a time period sufficiently long to renderthe virus non-infectious but less than that which would result indegradation of its antigenic characteristics, wherein said exposure isperformed in the substantial absence of oxygen and other oxidizingspecies.
 2. A method as in claim 1, wherein the furocoumarin is added toan inactivation medium containing the live virus.
 3. A method as inclaim 1, wherein the furocoumarin is introduced to the live virus byaddition to a cell culture medium in which the virus is grown.
 4. Amethod as in claim 1, wherein the inactivation medium is maintainedunder a non-oxidizing gas atmosphere.
 5. A method as in claim 4, whereinthe inactivation medium is flushed with the non-oxidizing gas.
 6. Amethod as in claim 4, wherein the non-oxidizing gas is selected from thegroup consisting of hydrogen, nitrogen, argon, helium, neon, carbondioxide, and mixtures thereof.
 7. A method as in claim 1, wherein anoxygen scavenger is added to the inactivation medium.
 8. A method as inclaim 7, wherein the oxygen scavenger is sodium ascorbate.
 9. Animproved method for inactivating viruses, said method being of the typewherein the virus is inactivated by exposure to long wavelengthultraviolet radiation in the presence of an inactivating furocoumarin,said improvement comprising performing said exposure to ultravioletradiation in the substantial absence of oxygen and other oxidizingspecies.